Sample was collected from rhizospheric soil. Among this study, two bacteria and two fungi were selected for siderophore production. The siderophore producing organisms were identified based on cultural, morphological and biochemical characteristics. Hence, the isolated colonies were confirmed as E.coli, Pseudomonas fluorescence, Rhizopus sp and Aspergillus flavus. By this study, analysis and nature of siderophore production was determined using these organisms. Pseudomonas fluorescens and Rhizopus sp showed the maximum catechol type of siderophore produced followed by E.coli and A.flavus minimum catechol type of siderophore. Among these, Pseudomonas fluorescens and Aspergillus flavus showed the maximum hydroxamate siderophore, followed by Rhizopus and E.coli minimum hydroxamate type of siderophore produced. Whereas, the optimization of medium for siderophore production maximum in bacteria when compared with fungi. The antibacterial activity was performed by well diffusion assay against clinical bacterial pathogens such as Streptococcus mutans, Salmonella typhi, Staphylococcus aureus and Klebsiella pneumoniae. High frequency of antibacterial activity was observed in Pseudomonas fluorescens are more efficient than the fungi Aspergillus flavus. Thus we concluded that rhizosphere soil organism producing siderophore to improve the plant growth and good antibacterial activity for clinical pathogens.